During prior funding periods, we have produced, crystallized and determined the structure of human (h) CD4 sCD41-183 and HIV-gp41 to high resolution. The structure of hsCD4 in complex with an MHC class II molecule (hsSCD4-pMHCII) was also solved. In conjunction with the hsCD4-HIV-1 gpl20-17b Fab complex, the information offers structural insight into how the AIDS virus has morphed its envelope protein to usurp the normal pMHCII binding site on CD4. Recent advances now position us to develop subunit vaccines potentially capable of eliciting broad neutralizing antibody (Nab) responses. These include 1) systems to express trimeric HIV-1 and SIV envelope ectodomains (gp140) capable of undergoing physiologic CD4-induced conformational change; 2) knowledge on the biophysical characteristics of Nab; 3) identification of critical site-specific local requirements for NAb binding; and 4) informatic analysis regarding T and B cell epitopes in HIV-I gpl60 proteins, suggesting relevant "mini-protein" constructs. Here, five groups of investigators will utilize their collective talents in structural immunology, crystallography, NMR, cryoelectron microscopy and vaccinology to pursue structure-based subunit vaccine design. The Reinherz/Wang group will continue X-ray crystallographic efforts on Lee3.2.8.1 produced Mac32H gp140 and derivative trimeric fragments as well as exploiting "miniprotein" designs including fusion to protein G B1 domain, C3d or Blys proteins in E. coli and Pichia, among other expression systems. The Harrison group will develop cryoelectron microscopy methodologies to investigate the entire gpl40 envelope and Nab interactions, complement X-ray efforts and offer structural insight for mini-protein design. The Wagner group will use large protein NMR methodology to confirm the native structure of engineered mini-proteins, providing guidance on ways to append solubilizing domains to function as molecular adjuvants. Wagner will also employ fluorescence polarization assays to identify HIV gpl40-binding organic inhibitors of the CD4-gpl40 interaction. IOMAI Corporation will use skin immunization technology to develop patch vaccine products for topical delivery of HIV envelope proteins. This strategy of provoking dual systemic and mucosal NAbs will be compared with other immunization methods in the Immunization and Neutralization Core to be headed by the Montefiori/Haynes group.